Project summary Proper control of gene expression is essential for maintenance of cell and tissue homeostasis. Altered regulation of gene expression contributes to a number of diseases including cancer and neurodegenerative and metabolic disorders. One important layer of gene regulation occurs at the post-transcriptional level and is orchestrated by a class of small non-coding RNAs called microRNAs (miRNAs). Alterations in miRNA function contribute to many diseases, yet we lack complete knowledge of how miRNA activity is controlled to differentially repress target genes. The broad goal of the proposed research program is to understand how miRNA activity is regulated in the complex context of a living organism. More specifically, we will address two questions that have limited our understanding of miRNA gene regulatory activity in development and disease. 1. How is miRNA target gene specificity established through regulation of miRNA induced silencing complex (miRISC) programming with a specific miRNA strand? and 2. How do auxiliary miRISC protein cofactors impact the gene-regulatory outcomes of miRNA activity on target mRNAs? To address these questions, we will take advantage of the powerful model organism Caenorhabditis elegans and utilize a number of approaches to define the genetic and molecular networks that regulate miRNA activity in vivo. The proposed work will build a foundation that will help us understand how misregulation of miRNA function contributes to loss of gene expression control in a variety of diseases.